Jing Hongyang, Chen Peng, Hui Tiankun, Yu Zheng, Zhou Jin, Fei Erkang, Wang Shunqi, Ren Dongyan, Lai Xinsheng, Li Baoming
School of Life Science, Nanchang University, Nanchang, 330031, China.
Institute of Life Science, Nanchang University, Nanchang, 330031, China.
Cell Biosci. 2021 Jun 5;11(1):105. doi: 10.1186/s13578-021-00619-z.
The neuromuscular junction (NMJ) is a peripheral synapse critical to muscle contraction. Like acetylcholine receptors (AChRs), many essential proteins of NMJ are extremely concentrated at the postjunctional membrane. However, the mechanisms of synapse-specific concentration are not well understood; furthermore, it is unclear whether signaling molecules critical to NMJ formation and maintenance are also locally transcribed.
We studied the β-gal activity encoded by a lacZ cassette driven by the promoter of the Lrp4 gene. As reported for Lrp4 mRNA, β-gal was in the central region in embryonic muscles and at the NMJ after its formation. However, β-gal was no longer in the central areas of muscle fibers in Lrp4 or MuSK mutant mice, indicating a requirement of Lrp4/MuSK signaling. This phenotype could be rescued by transgenic expression of LRP4 with a transmembrane domain but not soluble ECD in Lrp4 mutant mice. β-gal and AChR clusters were distributed in a broader region in lacZ/ECD than that of heterozygous lacZ/+ mice, indicating an important role of the transmembrane domain in Lrp4 signaling. Synaptic β-gal activity became diffused after denervation or treatment with µ-conotoxin, despite its mRNA was increased, indicating synaptic Lrp4 mRNA enrichment requires muscle activity. β-gal was also diffused in aged mice but became re-concentrated after muscle stimulation. Finally, Lrp4 mRNA was increased in C2C12 myotubes by Wnt ligands in a manner that could be inhibited by RKI-1447, an inhibitor of ROCK in Wnt non-canonical signaling. Injecting RKI-1447 into muscles of adult mice diminished Lrp4 synaptic expression.
This study demonstrates that synapse-specific enrichment of Lrp4 mRNA requires a coordinated interaction between Lrp4/MuSK signaling, muscle activity, and Wnt non-canonical signaling. Thus, the study provides a new mechanism for Lrp4 mRNA enrichment. It also provides a potential target for the treatment of NMJ aging and other NMJ-related diseases.
神经肌肉接头（NMJ）是对肌肉收缩至关重要的外周突触。与乙酰胆碱受体（AChR）一样，NMJ的许多重要蛋白质都极度集中于突触后膜。然而，突触特异性集中的机制尚未完全明确；此外，对于NMJ形成和维持至关重要的信号分子是否也在局部转录尚不清楚。
我们研究了由Lrp4基因启动子驱动的lacZ盒编码的β-半乳糖苷酶活性。正如Lrp4 mRNA的报道，β-半乳糖苷酶在胚胎肌肉的中央区域以及形成后的NMJ处。然而，在Lrp4或MuSK突变小鼠中，β-半乳糖苷酶不再存在于肌纤维的中央区域，这表明需要Lrp4/MuSK信号传导。在Lrp4突变小鼠中，具有跨膜结构域的LRP4的转基因表达可以挽救这种表型，但可溶性胞外结构域则不能。与杂合的lacZ/+小鼠相比，β-半乳糖苷酶和AChR簇在lacZ/ECD中的分布区域更广泛，表明跨膜结构域在Lrp4信号传导中起重要作用。去神经支配或用μ-芋螺毒素处理后，突触β-半乳糖苷酶活性变得弥散，尽管其mRNA增加，这表明突触Lrp4 mRNA的富集需要肌肉活动。β-半乳糖苷酶在衰老小鼠中也呈弥散状态，但在肌肉刺激后重新集中。最后，Wnt配体以一种可被RKI-1447（Wnt非经典信号传导中ROCK的抑制剂）抑制的方式增加了C2C12肌管中Lrp4 mRNA的表达。将RKI-1447注射到成年小鼠的肌肉中会减少Lrp4的突触表达。
本研究表明，Lrp4 mRNA的突触特异性富集需要Lrp4/MuSK信号传导、肌肉活动和Wnt非经典信号传导之间的协同相互作用。因此，该研究为Lrp4 mRNA富集提供了一种新机制。它还为治疗NMJ衰老和其他NMJ相关疾病提供了一个潜在靶点。
